During the past decade, there has been a tremendous growth in the use of proteins, such as monoclonal antibodies, for drug development and therapeutic applications. The production of pharmaceutical-grade monoclonal antibodies in large-scale, however, is a complex process requiring multiple chromatography and filtration steps to satisfy stringent regulatory requirements.
The manufacturing of biomolecules begins with the synthesis of the desired biological material in a biologically derived system. Preparation of the desired material is typically accomplished through a specifically designed and ordered series of physical separation techniques.
The manufacturing process for monoclonal antibodies, for example, typically includes expression in recombinant mammalian cell cultures. After centrifugation, the culture product is subjected to downstream purification (DSP) which generally comprises a “capture” stage, an “intermediate purification” stage and a “polishing” stage. The capture stage is usually based on Protein A affinity chromatography. The resulting product is then further purified and polished, typically using cation exchange and anion exchange chromatography steps to remove degradation products and impurities. Finally, viral species are removed through an included filtration step.
Protein A affinity chromatography, however, suffers from several disadvantages, including protein A leakage, and a simplified purification process has therefore been developed where a cation exchange capture stage replaces the protein A affinity chromatography. The cation capture step then removes process-related contaminants to such a low level that a single polishing step is enough to clean the residuals. Such a down stream purification of process monoclonal antibody thus includes a cation exchange chromatography step, an anion exchange chromatography step, and a virus removal filtration step.
The hitherto used down stream purification processes are performed with batch processes using hold-up tanks between the different chromatography and filtration steps. Each process step uses buffer or buffers of a certain pH and conductivity as well as post-process cleaning and storage liquids. Prior to each step, it is often necessary to adjust the conditions such as pH and protein concentration of the product stream, these adjustment steps then being performed as separate intermediate batch operations.
Separation systems and processes which to at least some extent allow for automation and on-line adjustment of eluates have been proposed in the prior art.
US 2009-0149638 discloses systems and processes for downstream protein purification on a large scale and which allow for automation, the processes being capable of being operated on a high-throughput and continuous basis. In the processes, one chromatography step follows another without an intermediate ultrafiltration/diafiltration step, the eluate from one tank being transferred to another via intermediate holding tanks which can be rendered acidic or basic.
US 2009-0126466 discloses multi-dimensional high performance liquid chromatography (HPLC) wherein a first chromatographic separation is performed at a first pH with a first mobile phase, and the fraction or fractions collected therefrom are subjected to a second chromatographic separation at a second different pH with a second mobile phase. The fraction or fractions collected from the first separation may be concentrated or diluted, optionally on-line, prior to subjecting the fraction or fractions to the second chromatographic separation mode.
It is an object of the present invention to provide an improved automated separation system which is capable of further reducing development time, processing time and cost of goods and which does not require intermediate holding tanks.